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| United States Patent |
5,789,179
|
|
Brophy
, et al.
|
August 4, 1998
|
Cat assay
Abstract
A method of assaying for CAT in a fluid involves the use of a complex of
chloramphenicol with a member of a specific binding pair such as a hapten
or biotin. Biotinylated chloramphenicol is claimed as new. A scintillation
proximity assay involves use of this reagent with tritiated acetyl
coenzyme A and streptavidin coated SPA beads.
| Inventors:
|
Brophy; Gerard Philip (Cardiff, GB);
Cummins; William Jonathan (Tring, GB);
Mundy; Christopher Robert (Cambridge, GB)
|
| Assignee:
|
Amersham International plc (GB)
|
| Appl. No.:
|
861536 |
| Filed:
|
May 22, 1997 |
Foreign Application Priority Data
| Current U.S. Class: |
435/7.4; 435/7.5; 435/7.7; 435/15; 435/964; 436/518; 436/523; 436/524; 436/525; 436/526; 436/527; 436/528; 436/529; 436/530; 436/531; 436/532; 436/533; 436/534; 436/536; 436/542; 436/804 |
| Intern'l Class: |
G01N 033/573; G01N 033/53 |
| Field of Search: |
435/7.4,7.5,7.7,15,964
436/518,523-534,536,542,804
|
References Cited
U.S. Patent Documents
| 4489156 | Dec., 1984 | Khanna et al.
| |
| 4489157 | Dec., 1984 | Khanna et al.
| |
| 4568649 | Feb., 1986 | Bertoglio-Matte | 436/534.
|
| 5262545 | Nov., 1993 | Haughland et al. | 548/405.
|
| Foreign Patent Documents |
| 0387875A3 | Sep., 1990 | EP.
| |
| 2312780 | Jan., 1977 | FR.
| |
| WO8903042 | Apr., 1989 | WO.
| |
Other References
Derwent Abstracts, Database WPI, Week 9127, Derwent Publications Ltd.,
London, GB; AN91-198111 (1991).
Chemical Abstracts, 90:106x (1979).
Sigma Catalog p. 1122, 1993.
|
Primary Examiner: Huff; Sheela
Attorney, Agent or Firm: Wenderoth, Lind & Ponack, L.L.P.
Parent Case Text
This application is a continuation of application, Ser. No. 08/334,228,
filed Nov. 4, 1994, now abandoned.
Claims
We claim:
1. A method of assaying for CAT in a fluid, which method comprises
incubating in the presence of a sample of the fluid; a biotinylated
chloramphenicol which comprises a member of a specific binding pair; a
radiolabelled acyl coenzyme A; and a third reagent which is the other
member of the specific binding pair, and is bound to a solid support,
wherein there is formed a complex;
streptavidin or avidin--(chloramphenicol)--(acyl group) to an extent
related to the CAT activity of the fluid sample, said complex being bound
to the said solid support.
2. The method as claimed in claim 1, wherein the acyl coenzyme A is
radioactively labelled acetyl coenzyme A.
3. The method as claimed in claim 1, wherein the acyl coenzyme A is acetyl
coenzyme A labelled with tritium, and the third reagent is immobilized on
a solid support.
4. The method as claimed in claim 1, wherein the solid support is a
scintillation proximity assay support.
5. The method as claimed in claim 1, wherein the chloramphenicol analogue
is (+)-D-threo-6-biotinylamino-N›(2-hydroxy-1-hydroxymethyl)-2-(4-nitrophe
nyl) ethyl!-hexamide.
Description
BACKGROUND OF THE INVENTION
Chloramphenicol Acetyl Transferase (CAT) is the most commonly used reporter
gene in molecular biology. The enzyme catalyses the reaction of
chloramphenicol and acetyl coenzyme A to produce acetylated
chloramphenicol. The enzyme has no eukaryotic counterpart and therefore is
a useful measure of transfection efficiency and translational activity in
cultured cells. Typically the gene, contained on a plasmid, is transfected
into the cell of interest and, after an appropriate incubation period, CAT
activity is measured.
CAT has a number of advantages for use as a reporter gene:
The enzyme activity is readily distinguishable from that of endogenous
cellular proteins present in the cell prior to transfection.
There is no interference or competition from other enzymatic activities in
the cells.
CAT is a relatively stable enzyme exhibiting a degree of thermotolerance
and a lack of inhibition by cellular components.
PRESENT METHODS OF CAT ACTIVITY DETERMINATION
.sup.14 C Chloramphenicol TLC
This is the most common method of assaying for CAT activity. The cell
extract containing CAT is incubated with .sup.14 C chloramphenicol and
cold acetyl coA. After incubation the reactants are extracted into an
organic solvent and dried down. The reactants are resuspended in a small
volume and spotted on Thin Layer Chromatography (TLC) plates which are
developed to separate chloramphenicol from its acetylated derivatives. The
plates are then exposed to autoradiographic film to locate the radioactive
spots. These can then be scraped off, extracted and scintillation counted
to give a measure of acetylation activity. Alternatively if a radioactive
imaging system, such as a phosphorimager, is available the plates can be
exposed and directly quantificated by this means.
The disadvantages of this method are as follows:
The assay is cumbersome and time consuming.
The assay involves TLC.
Organic solvents are used.
Quantification is difficult.
Sensitivity is limited.
It is difficult to assay large numbers of samples.
Labelled acetyl coA
A variation on the above assay uses a radioactive acetyl group on the coA
and cold chloramphenicol. After the reaction the mix is extracted with
organic solvent or overlayed with water immiscible scintillation fluid.
Acetylated forms of chloramphenicol diffuse into the organic phase and can
be counted.
Disadvantages
The assay is difficult to perform and often yields unsatisfactory results
due to phase flipping problems.
Fluorescent chloramphenicol
A chloramphenicol with a fluorescent molecule attached is commercially
available. This assay format is carried out by adding this fluorescent
chloramphenicol and acetyl coA to the cell extract. The chloramphenicol
and its reaction products are separated by TLC and visualised by UV
illumination. The spots can be scraped off and quantificated in a
fluorimeter.
Disadvantages
The assay is cumbersome and time consuming.
The assay requires a TLC separation step.
Sensitivity is low.
A fluorimeter may not be easily accessible in every laboratory.
CAT ELISA
A CAT ELISA kit is commercially available. In this assay format cell
extract is pipetted into MTP wells which contain bound CAT antibody. The
wells are washed and incubated with anti-CAT digoxygenin. Again the wells
are washed and incubated with anti-digoxygenin peroxidase. The wells are
washed and incubated with a peroxidase substrate yielding a colourimetric
endpoint which can be quantificated on an MTP reader.
Disadvantages
CAT presence not activity is measured.
The assay is relatively time consuming due to the many washing steps.
Sensitivity is low.
SUMMARY OF THE INVENTION
This invention provides an improved method of assaying for CAT, based on
the use of chloramphenicol analogues, some of which are also claimed
herein as new compounds.
In one aspect the invention provides a method of assaying for CAT in a
fluid, which method comprises incubating in the presence of a sample of
the fluid, a chloramphenicol analogue which comprises a member of a
specific binding pair, a radiolabelled acyl coenzyme A analogue, and a
third reagent which comprises the other member of the specific binding
pair, whereby there is formed a complex
(third reagent)--(chloramphenicol analogue)--(acyl group)
to an extent related to the CAT activity of the fluid sample.
A chloramphenicol analogue is a compound, related to chloramphenicol, which
is capable of reacting with acetyl coenzyme A, in a reaction catalysed by
CAT, to give the 3-acetyl derivative. The analogue is a modified
chloramphenicol derivative, which may for example have lost its
dichloroacetyl function. An analogue may be chemically identical to the
originating compound; for example, a radiolabelled compound is regarded
herein as being an analogue of the unlabelled compound.
Specific binding pairs are well known in biochemistry. They include, but
are not limited to: antigen/antibody; hapten/antibody; biotin/avidin;
biotin/streptavidin; single stranded DNA/complementary DNA.
The chloramphenicol analogue comprises a member of a specific binding pair,
and may be a conjugate of chloramphenicol with the member of the specific
binding pair, the conjugate having the structure shown
##STR1##
where R.sup.1 is --NO.sub.2 or --X-Z,
R.sup.2 is --NHCOR.sup.3 or --X-Z,
provided that at least one of R.sup.1 and R.sup.2 is --X-Z,
R.sup.3 is CHCl.sub.2 or C.sub.1 -C.sub.6 alkyl,
Z is a member of a specific binding pair, e.g. a hapten, and
X is a spacer arm of sufficient length to enable the hapten or other member
of a specific binding pair to bind correctly with its specific binding
partner.
When R.sup.1 is NO.sub.2 and R.sup.2 is NHCOCHCl.sub.2, the compound is
chloramphenicol. The two asymmetric carbon atoms generate four possible
stereoisomers, of which only the D-threo isomer has significant biological
activity. Preferred chloramphenicol analogues are based on this
stereoisomer.
Z may be a hapten such as biotin, fluorescein or digoxygenin. The spacer
arm X may typically be from 2 to 25 carbon atoms, preferably 6 carbon
atoms in length. Certain of these chloramphenicol analogues are new
compounds, and are included as such in this invention. Among these are
compounds having the formula 1 above in which
either R.sup.1 is NO.sub.2 and R.sup.2 is X-biotin,
or R.sup.1 is X-biotin and R.sup.2 is NHCOCH.sub.3,
and X is a spacer arm of sufficient length to enable the biotin moiety to
bind correctly with its specific binding partner. Preferably X contains 6
carbon atoms, so that the compound is either
(+)-D-threo-6-biotinylamino-N›(2-hydroxy-1-hydroxymethyl)-2-(4-nitrophenyl
) ethyl!-hexamide; or
6-biotinylamino-N›4-(N-acetyl-1-(2-amino-1,3-propandiol))phenyl! hexamide.
An acyl coenzyme A analogue is a compound, related to acetyl coenzyme A,
which is capable of undergoing a reaction with chloramphenicol, catalysed
by CAT, resulting in the production of a 3-acyl derivative of
chloramphenicol. The acyl coenzyme A analogue may be acetyl coenzyme A,
radiolabelled with e.g. tritium or carbon-14. Or the acyl coenzyme A
analogue may be an acyl coenzyme A, in which the acyl is e.g. propionyl or
butyryl. The acyl group of the acyl coenzyme A analogue is radiolabelled.
The nature of the radiolabel is not material to the invention.
The method of the invention involves the use of a third reagent which is or
comprises a member of a specific binding pair; the other member of this
specific binding pair is comprised in the chloramphenicol analogue. For
example, when the biotinylated chloramphenicol analogue is used, the third
reagent may be avidin or streptavidin.
The chloramphenicol analogue, the acyl coenzyme A analogue and the third
reagent are incubated, in the presence of a sample of the fluid under
test, in order to form a complex
(third reagent)--(chloramphenicol analogue)--(acyl group).
In this complex, the acyl group is radiolabelled. In order to facilitate
separation of the complex from unreacted label, the third reagent may be
bound to a solid support, for example to a wall of an assay vessel or to
solid particles suspended in the assay medium. The assay reagents may be
incubated all together, or alternatively the chloramphenicol analogue may
be pre-reacted with the third reagent.
There follow examples of preferred assay formats according to the
invention. These involve the use of chloramphenicol-hapten conjugates as
the chloramphenicol analogue. The hapten is used for separation/attachment
purposes and the radiolabelled acyl group provides the means of detection.
A. SPA-CAT Assay
SPA, the Scintillation Proximity Assay is covered by U.S. Pat. No.
4,568,649, European Patent No. 0154734 and Japanese Patent Application No.
84/52452.
In this assay format a haptenated chloramphenicol analogue (the hapten is
preferably biotin) is incubated with tritiated (.sup.3 H) acetyl coenzyme
A. Suitably coated SPA beads (e.g. streptavidin or specific antibodies)
are added and bind to the acetylated, haptenated chloramphenicol analogue
produced giving rise to a scintillation signal which is a measure of
enzyme activity.
The assay is quick and easy to use. Results are possible in one hour after
extract production. No separation steps are required and the entire assay
procedure can be carried out in one tube.
In addition the assay is very sensitive (at least fifteen fold more
sensitive than .sup.14 C chloramphenicol TLC assays).
B. CAT assay with coated beads
In this assay format tritiated, acetylated, haptenated chloramphenicol
analogue is produced as before in a microcentrifuge tube. Suitably coated
beads are added which bind to the haptenated molecule. The beads are
centrifuged to form a pellet and the supernatant, which contains the
unreacted tritiated acetyl coenzyme A, is removed. The pellet is washed
and scintillation fluid added. The scintillation signal produced is a
measure of CAT activity.
The assay, although not homogeneous, requires only a single separation step
and can be carried out in one tube. It is therefore quicker and easier and
more sensitive than existing CAT assays.
BRIEF DESCRIPTION OF THE DRAWINGS
Reference is directed to the accompanying drawings in which:
FIG. 1 is a diagram showing the synthesis of
(+)-D-threo-6-biotinylamino-N›(2-hydroxy-1-hydroxymethyl)-2-(4-nitrophenyl
)ethyl!-hexanamide (Compound 1).
FIG. 2 is a mass spectrometry trace of Compound 1.
FIG. 3 is a diagram showing the synthesis of
6-biotinylamino-N›4-(N-acetyl-1-(2-amino-1,3-propandiol))phenyl!hexamide
(Compound 2).
The following examples illustrate the invention.
DETAILED DESCRIPTION
General Details
Biotinamidocaproic acid N-hydroxysuccinimide ester was obtained from Sigma.
D-threo-2-amino-1-(4-nitrophenyl)-1,3-propandiol was obtained from Aldrich
or produced by base hydrolysis of chloramphenicol. Triethylamine was
predistilled from sodium hydroxide. Anhydrous solvents refer to Aldrich
Sure Seal bottled material. Other solvents and reagents are available from
several commercial sources.
.sup.1 H NMRs were obtained on a Jeol 270 MHz machine using d.sub.4 MeOH as
solvent.
TLCs were run on Kieselgel 60 F.sub.254 aluminium backed TLC plates using
the solvent system quoted.
EXAMPLE 1
Synthesis of
(+)-D-threo-6-biotinylamino-N›2-hydroxy-1-(hydroxymethyl)-2-(4-nitrophenyl
)ethyl!-hexamide.
(Compound 1)
The synthetic reaction sequence is shown in FIG. 1.
In a clean, round-bottom flask biotinamidocaproic acid N-hydroxysuccinimide
ester (230 mg 0.51 mmoles) was dissolved in anhydrous dimethylformamide (4
ml). To this was added D-threo-2-amino-1-(4-nitrophenyl)-1,3-propandiol
(110 mg 0.52 mmoles) and triethylamine (50 .mu.l). The flask was then
stoppered and the contents stirred overnight at room temperature.
Product formation was verified by pipetting 0.5 .mu.l of the reaction onto
a TLC plate and developing in chloroform/methanol 9:1. The product has an
R.sub.f of 0.2.
The product was purified using a preparative scale reverse phase HPLC
column (Hamilton PRP-1 preparative column 21:4 mm id.times.250 mm). An
acetonitrile/water gradient elution was used at a flow rate of 8 ml/min.
Gradient profile
______________________________________
Time (min) % acetonitrile
______________________________________
0 10
40 50
45 100
50 10
______________________________________
The product peak's retention time was approximately 30 mins as measured by
UV absorption at 254 nm. The solvent from the product fraction was rotary
evaporated off and the product was resuspended in ethyl acetate (5 ml).
The white solid was filtered on a sintered glass funnel and then air
dried. The solid was placed under high vacuum for one hour to remove the
last traces of solvent. Yield 200 mg (71%). The melting point was
determined as 173.degree. C.-175.degree. C. 270 MHz .sup.1 H NMR d.sub.4
MeOH .delta.: 1.1-1.8 (m, 12H), 2.1 (t, 2H), 2.7 (d, 1H), 2.9 (m, 1H), 3.1
(m, 2H), 3.2 (m, 1H), 3.5 (m, 1H), 3.7 (m, 1H), 4.1 (m, 1H), 4.3 (m, 1H),
4.5 (m, 1H), 5.1 (d, 1H), 7.6 (d, 2H), 8.2 (d, 2H).
The product was analysed by mass spectrometry. A trace is provided in FIG.
2.
A kinetic analysis of CAT and this biotinylated chloramphenicol analogue
was carried out to characterise this new substrate. This work determined
that the enzyme's Km for chloramphenicol was 11.0 .mu.M and for the
biotinylated chloramphenicol analogue was 9.5 .mu.M. The kcats were 97
s.sup.-1 and 93 s.sup.-1 respectively. These data indicate that the
biotinylated chloramphenicol analogue is a suitable substrate for CAT and
that the reaction rates of the enzyme are similar with both substrates.
EXAMPLE 2
Standard curve of Chloramphenicol Acetyl Transferase (CAT) activity.
In this assay the biotinylated chloramphenicol analogue of Example 1 is
acetylated by CAT using ›.sup.3 H! acetyl coenzyme A as the acyl donor.
The tritiated acetylated biotinylated chloramphenicol analogue binds to
Scintillation Proximity Assay (SPA) beads and gives rise to a
scintillation signal which can be related to enzyme activity.
Materials and Methods
CAT enzyme obtained from Professor W. V. Shaw, Biochemistry Department,
Leicester University, was diluted to concentrations of 0.02, 0.04, 0.06,
0.08 and 0.1 units of enzyme with 0.1M Tris/HCl (pH 7.8) in total volume
of 40 .mu.l. One unit of enzyme is the amount which catalyses the
acetylation of one nanomole of chloramphenicol in one minute at 37.degree.
C. Duplicate samples were assayed. To each assay tube was added 10 .mu.l
of a master mix which contained:
______________________________________
biotinylated chloramphenicol analogue (0.1 mmolar)
1 .mu.l
›.sup.3 H! acetyl coenzyme A (Amersham TRK688)
2 .mu.l (0.5 .mu.Ci)
water 1 .mu.l
Tris/HCl, pH 7.8, 1 molar
6 .mu.l.
______________________________________
The reaction mixture was mixed and incubated at 37.degree. C. for 30
minutes.
After incubation 0.5 mg of streptavidin coated SPA beads (Amersham) were
added in 0.75 ml PBS. The reaction tubes were placed in scintillation
vials and counted.
The results are tabulated below.
______________________________________
CAT (units)
cpm
______________________________________
0 181
197
0.02 11304
11485
0.04 24026
23873
0.06 34921
34957
0.08 46748
45050
0.1 50846
52762
______________________________________
As can be seen the increase in CAT activity is proportional to the increase
in cpm.
EXAMPLE 3
Synthesis of
6-biotinylamino-N›4-(N-acetyl-1-(2-amino-1,3-propandiol))phenyl!hexamide
(Compound 2).
The synthetic reaction sequence is shown in FIG. 3.
The synthetic details for each intermediate compound in the synthetic
sequence and for the final synthetic step are set out below.
N-›2-hydroxy-1-(hydroxymethyl)-2-(4-nitrophenyl)ethyl! acetamide.
Intermediate 1
In a clean, round bottom flask
D-threo-2-amino-1-(4-nitrophenyl)-1,3-propandiol (1.43 g, 6.75 mmoles) was
suspended in anhydrous tetrahydrofuran (30 ml). To this was added acetic
anhydride (0.8 ml, 8.44 mmoles). The flask was stoppered and the contents
stirred at room temperature for one hour.
Product formation was verified by spotting a small aliquot of the reaction
mixture onto a TLC plate and developing in ethyl acetate/methanol 9:1. The
product has an R.sub.f of 0.33 and is negative to a ninhydrin spray.
The tetrahydrofuran was removed on a rotary evaporator and the resulting
gummy residue subjected to a silica flash column to purify the product.
The eluant for the column was ethyl acetate/methanol 9:1. The product
fractions were pooled and the solvent removed on a rotary evaporator. The
resulting foam was placed under high vacuum for one hour to remove the
last trace of solvent. The yield was 1.28 g (78%). 270 MHz .sup.1 H NMR
d.sub.4 MeOH .delta.: 1.8 (s, 3H), 3.5 (m, 1H), 3.7 (m, 1H), 4.1 (m, 1H),
5.1 (brs, 1H), 7.6 (d, 2H), 8.2 (d, 2H).
N-›2-hydroxy-1-(hydroxymethyl)-2-(4-aminophenyl)ethyl! acetamide.
Intermediate 2
In a clean, round bottom flask
N-›2-hydroxy-1-(hydroxymethyl)-2-(4-nitrophenyl)ethyl! acetamide (1.27 g 5
mmoles) was dissolved in methanol (50 ml). The flask was then fitted with
a suitable adaptor and coupled to an hydrogenation apparatus. The
atmosphere in the flask was then replaced with nitrogen. The adaptor was
then removed and 10% palladium on carbon catalyst (466 mg) was added to
the flask. The adaptor was replaced and the hydrogenation apparatus
manipulated to replace the atmosphere in the flask with hydrogen. The
flask contents were then stirred at room temperature under an atmosphere
of hydrogen overnight. The hydrogenation apparatus was manipulated to
replace the atmosphere in the flask with nitrogen. The flask was then
disconnected from the hydrogenation apparatus.
The palladium on carbon catalyst was removed by filtering through a hyflo
filter pad. The solvent from the product was rotary evaporated off to give
a gum. The gum was placed under high vacuum for one hour to remove the
last traces of solvent. The yield of product was 1.05 g (94%). 270 MHz
.sup.1 H NMR d.sub.4 MeOH .delta.: 1.9 (s, 3H), 3.4 (m, 1H), 3.6 (m, 1H),
4.0 (m, 1H), 4.7 (d, 1H), 6.7 (d, 2H), 7.1 (d, 2H).
The Intermediate 2 product was used in the next synthetic step without any
purification.
6-biotinylamino-N›4-(N acetyl-1-(2 amino-1,3-propandiol))phenyl!hexamide
Biotinamidocaproic acid N-hydroxysuccinimide ester (100 mg 0.22 mmoles) was
weighed out into a clean, round bottomed flask. A solution of
N-›2-hydroxy-1-(hydroxymethyl)-2-(4-aminophenyl)ethyl! acetamide (108 mg,
0.51 mmoles) in anhydrous dimethylformamide (2 ml) was added to the flask.
Triethylamine (25 .mu.l) was added to the flask which was then stoppered.
The reaction mixture was stirred at room temperature for a total of 80
hours and at 80.degree. C. for a total of 6 hours.
The major product was then purified using a preparative scale reverse phase
HPLC column (Hamilton PRP-1 preparative column 21.4 mm id.times.250 mm).
An acetonitrile/ammonia solution (2ml of 0.880 NH.sub.3 /litre of water)
gradient elution was used at a flow rate of 8 ml/min.
Gradient profile
______________________________________
Time (min) % acetonitrile
______________________________________
0 0
45 40
50 100
55 0
______________________________________
The product peak retention time was approximately 35 min as measured by UV
absorption at 254 mm. The solvent from the product fraction was rotary
evaporated off to give an off white solid, the yield was 24 mgs (20%). 270
MHz .sup.1 H NMR d.sub.4 MeOH .delta.: 1.2-1.7 (m, 12H), 1.9 (s, 3H), 2.1
(t, 2H), 2.3 (t, 2H), 2.6 (d, 1H), 2.8 (m, 1H), 3.1 (m, 3H), 3.4 (m, 1H),
3.5 (m, 1H), 4.0 (m, 1H), 4.3 (m, 1H), 4.4 (m, 1H), 7.2 (d, 2H), 7.4 (d,
2H).
EXAMPLE 4
6-biotinylamino-N›4-(N-acetyl-1-(2-amino-1,3-propandiol))phenyl!hexamide,
(Compound 2) is a substrate of CAT.
Biotinylated chloramphenicol analogue (compound 2) was dissolved to a
concentration of 2 mmolar in 50% methanol. .sup.14 C acetyl coenzyme A (50
.mu.Ci/ml, 58 mCi/mole) was obtained commercially.
In a microcentrifuge tube were mixed Tris/HCl (1M, pH 7.8) 7.5 .mu.l,
biotinylated chloramphenicol analogue (2 mM) 80 .mu.l, CAT enzyme 3.75
units and .sup.14 C acetyl coenzyme A 2 .mu.l (0.1 .mu.Ci). The mix was
incubated at 37.degree. C. for 90 minutes. A control reaction which
contained no enzyme was performed in parallel. 20 .mu.l of each reaction
was spotted on to a cellulose TLC plate which was developed in 95/5
chloroform methanol. The TLC plate was exposed to autoradiographic film.
The lane corresponding to the reaction to which enzyme was added contains a
spot which migrated under the influence of the solvent. This band is
acetylated, biotinylated chloramphenicol analogue. No corresponding band
is visible in the no enzyme control.
This experiment proves that 6-biotinylamino-N›4-(N
acetyl-1-(2-amino-1,3-propandiol))phenyl!hexamide is acetylated with
.sup.14 C acetyl coenzyme A by CAT.
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